Pain from cancer, particularly cancer that metastasizes to bone, is often severe, difficult to manage, and contributes significantly to the patients' poor quality of life. Although opioids remain the primary treatment for managing severe cancer pain, the relative resistance of cancer pain to opioids requires increased doses, which limit their use because of the many undesirable side effects associated with actions in the central nervous system. It is important to understand the mechanisms by which opioids become less effective in treating cancer pain. The mechanisms that drive cancer pain are multi-factorial and include bone remodeling, inflammatory responses, neurodegeneration, and release of algesic substances from the cancer cells that excite or sensitize nociceptors on primary afferent fibers. ATP is one of the known algesic substances that cancer cells contain and release during tumor growth. ATP activates peripheral P2X receptors which are located on nociceptive nerve endings. Previous behavioral studies from our group indicated that ATP contributes to the development of hyperalgesia in a murine model of bone cancer. Moreover, opioids modulate signaling at P2X receptors. Opioids such as morphine decrease ATP-evoked currents that occur through P2X receptors. We hypothesize that this opioid modulation of signaling through the P2X receptor is diminished by cancer cells, and that this may contribute to the decreased effect of opioids in cancer pain. In the proposed project, we will use an in vivo and a new in vitro co-culture method to determine the effect of cancer cells on ATP-evoked responses of dorsal root ganglion (DRG) neurons, how those responses are modulated by 5- and 4-opioid receptor agonists, and whether cancer cells promote a change in the expression of opioid receptors in DRG neurons. We will determine the effect of fibrosarcoma cells on P2X- dependent activation of mouse DRG neurons, changes in opioid modulation of P2X currents produced by cancer cells, and alterations in expression of 5- and 4-opioid receptors. Also, we will compare changes that occur in vivo (in tumor-bearing mice) to changes that occur in our co-culture model in vitro. The proposed studies will be done in Ukraine (Kiev) at the Bogomoletz Institute of Physiology in collaboration with Dr. Oleg A. Krishtal, who is an expert in patch clamp electrophysiology and P2X receptor function. This study is an extension of our ongoing project, NIH Grant CA091007, in which we are examining the contribution of peripheral P2X and TRPV1 receptors in cancer pain and nociceptor sensitization Results from these studies will provide new information on the mechanisms by which ATP contributes to cancer pain, and how opioids modulate ATP-evoked responses during tumor growth. Understanding how opioids can modulate cancer-related changes in responses of sensory neurons may lead to the development of novel approaches for managing cancer pain. PUBLIC HEALTH RELEVANCE: It is estimated by the National Cancer Institute that more than 1.4 million new cases of cancer were diagnosed in the United States in 2007, and the World Health Organization estimates that up to 15 million new cases of cancer may be diagnosed world-wide in 2020. Approximately 85% of adult patients with terminal cancer report intolerable pain and up to 75% of children with cancer experience pain. Understanding the mechanisms that drive cancer pain so that new and improved therapeutic approaches for pain management can be developed is a relevant public health issue.